【作者】 柯丹；

【导师】 刁庆春； 李发琪；

【作者基本信息】 重庆医科大学 ， 生物医学工程， 2007， 硕士

【摘要】 超声波被广泛应用于医学治疗和诊断领域,高强度聚焦超声作为一种无创性治疗手段正日益受到人们的重视。低强度脉冲超声波(low intensity pulsed ultrasound,LIPUS)具有刺激细胞增殖和细胞外基质合成增加的作用,已逐渐应用于骨与软组织创伤的愈合和神经修复治疗中。目前,LIPUS对人皮肤成纤维细胞的影响研究还较少,为了探讨超声治疗皮肤疾病的应用前景,我们研究了LIPUS对人皮肤成纤维细胞的增殖及胶原蛋白合成的影响,为LIPUS治疗皮肤疾病提供理论依据。并开展了聚焦超声治疗神经性皮炎和慢性湿疹的临床研究,进行了超声治疗皮肤疾病的初步探讨。一、LIPUS促进皮肤成纤维细胞增殖的适宜辐照参数筛选用辐照参数为1 MHz、1.2 MHz、1.7 MHz,每一频率下四种不同声强的LIPUS,同一声强分别辐照0 min(假照组)、5 min、10 min、15 min、20 min和30 min体外培养的成纤维细胞,辐照24 h后观察成纤维细的生长增殖情况,采用MTT法测定其吸光光度值,计算其相对成活值,通过比较不同辐照参数对细胞的相对成活率的影响,筛选出促进皮肤成纤维细胞生长的最适宜频率、声强和辐照时间。结果: (1)用MTT法测定三种频率(1 MHz、1.2 MHz、1.7 MHz),每一频率下四种声强的LIPUS辐照成纤维细胞5 min,24 h后吸光光度值均增加,辐照成纤维细胞10 min,24 h后吸光光度值增加最高,较对照组都有显著性差异(P<0.05)。超过一定辐照时间后,吸光光度值下降,且随着辐照时间的延长,吸光光度值下降显著。(2)比较细胞的相对成活率发现,频率1 MHz、声强0.1 W/cm2、辐照时间5 min时细胞相对成活率为1.195,10 min时达1.325,与其它辐照参数组比较差异有统计学意义(P<0.05),两者之间比较P<0.05,有统计学意义。10 min时的细胞相对成活率最高,与对照组比较差异有统计学意义(P<0.05)。结论:⑴不同频率、不同声强的LIPUS辐照一定时间均能促进人真皮成纤维细胞增殖,超过一定辐照时间则会抑制细胞生长。⑵频率1 MHz、声强0.1 W/cm2、辐照时间10 min为促进成纤维细胞增殖较为适宜的辐照参数。二、低强度脉冲超声对皮肤成纤维细胞增殖及胶原蛋白合成的影响采用最适宜声参数——频率1 MHz、声强0.1 W/cm2的LIPUS辐照体外培养的人皮肤成纤维细胞,分别辐照5 min,10 min及20 min,采用H3-胸腺嘧啶核苷掺入试验检测其对细胞合成DNA的影响,H3-脯胺酸掺入试验检测其对细胞合成胶原蛋白的影响,并用流式细胞仪检测其对细胞周期的影响。结果:频率1 MHz、声强0.1 W/cm2的LIPUS辐照成纤维细胞5 min,24 h后细胞H3-胸腺嘧啶核苷H3- TdR掺入量为对照组的1.27倍、H3-脯胺酸掺入量为对照组的2.22倍,辐照10 min,24 h后H3- TdR掺入量为对照组的1.55倍、H3-脯胺酸掺入量为对照组的2.31倍,与对照组比较均有统计学差异(P<0.05),而辐照10 min时掺入量达最高,较5 min组有显著性差异(P<0.05),随着辐照时间进一步增加掺入量反而下降。LIPUS辐照5 min,S期细胞为(13.84±1.77)%,较对照组增加(P<0.05);辐照10 min后S期细胞为(20.56±3.56)%,较对照组增加(P<0.05);而辐照10 min G2/M期细胞(15.89±1.75)%,较对照组下降(P<0.05)。结论:LIPUS辐照一定时间能够促进人皮肤成纤维细胞DNA及胶原蛋白合成增加,并可以使细胞的周期发生改变,促进细胞从G2/M期进入S期,从而促进细胞的增殖。三.聚焦超声治疗神经性皮炎和慢性湿疹的初步探讨采用自身对照法对聚焦超声治疗51例神经性皮炎患者和51例慢性湿疹患者进行了疗效观察和安全性评价。治疗组采用重庆海扶技术有限公司研制生产的CZP型皮肤超声治疗仪进行治疗,对照组治疗采用安慰剂(白凡士林)外涂,每日2次。治疗后一月观察病人的主观症状、体征以及出现的其他不良反应,评价超声治疗的有效性和安全性,并进行统计分析。结果:治疗后1月,治疗组神经性皮炎患者和慢性湿疹患者各项瘙痒/体征指标计分及总计分均有明显下降,与对照组组间比较有显著差异(P<0.05〉。治疗后1月,神经性皮炎患者治疗组的痊愈率和痊愈显效率分别为2%和52.9%;慢性湿疹患者治疗组的痊愈率和痊愈显效率分别为4%和54.9%,与对照组比较均显著性增加(P<0.05〉;不同程度(重度>9,轻度≤9)神经性皮炎和慢性湿疹治疗1月后疗效无显著性差异;超声治疗后患者瘙痒情况明显改善,治疗后90.2%慢性湿疹患者、80.4%神经性皮炎患者瘙痒基本消失或明显减轻;治疗后无不良反应发生。结论:超声波可以有效治疗慢性湿疹和神经性皮炎,是一种新型的、有效的、安全的物理治疗手段,是一种值得推广也需要进一步研究的新兴物理治疗方法。更多还原

【Abstract】 Ultrasound now is widely used both in diagnosis and in therapy. As a new noninvasive local therapy, high intensity focused ultrasound (HIFU) therapy is gaining recognition from more and more doctors and patients. Previous studies revealed that low intensity pulsed ultrasound (LIPUS) had specific biological effects. The results of experiments in vitro and in vivo suggested that low intensity pulsed ultrasound could influence cell proliferation, promote protein synthesis, influence cytokine release and induce cell differentiation. LIPUS is used to accelerate fracture healing, to treat osteoradionecrosis and to repair epidermis after wound. But experimental investigations into the biological effects of LIPUS on skin fibroblast are quite scarce.This study was designed to investigate the changes in the cellular proliferation activities and the ability of collagen synthesis after LIPUS stimulation and screen out the optimal parameters of LIPUS to promote skin fibroblast proliferation, and provide reference for clinical use. We also explore the efficacy and safety of focused ultrasound in treatment of chronic eczema and neurodermatitis.PART I Screening of the opimal LIPUS parameters for skin fibroblast proliferationLIPUS of different frequency(1, 1.2, 1.7MHz)and four different intensities of each frequency was used to sonication the cultured human fibroblast cells. Under each frequency and intensity, the cells were randomly divided into 6 groups according to ultrasound exposure duration: 0 minute (the sham group), 5 minutes,10 minutes,20 minutes and 30minutes. 24 hours later, cell proliferation was examined, and the relative survival rate was calculated using MTT assay. Each test was repeated three times. After a comparison of the relative survival rate, we screened out the optimal parameters.Results: (1) As compared with the control group(P<0.05), the optical absorption value of cell activity in MTT assay increased significantly in the different frequency groups(1、1.2、1.7MHz) for 5and 10 minutes and the peak time is 10 imnutes . But the optical absorption value decreased with longer sonication time and the value decreased significantly in human skin fibroblasts with LIPUS sonication for more than 30 minutes (P<0.05). (2) The relative survival rate of the group 1MHz,0.1W/cm2 and 5 minutes was 1.195,and the 10 minutes group was 1.325; the peak relative survival rate was detected in the group with the LIPUS of 1MHz,0.1W/cm2 sonication for 10 minutes . 10004.22±743.53, which was 2.31 times of the sham group. A peak value was detected in the cell culture with LIPUS sonication for 10 minutes.But with longer sonication time, the value decreased.(2) The cell number in the S phase of the 5 minutes group was (13.84±1.77)%, which was a significant higher than the sham group(P<0.05); and the cell number in the G2/M phase was (19.64±2.88)%, which was reduced compared with the sham group. The peak changes were detected in the 10 minute group, where the cell number of the S phase was (20.56±3.56)% and the cell number in the G /M phase was (15.89±1.75)%. Both were significantly different as compared with the sham group.Conclusions: Sonication with LIPUS of 1MHz and 0.1W/cm2 in a limited time could promote DNA synthesis and collagen production of skin fibroblasts, and promote cell transition from G2/M phase to S phase, so fibroblast cell proliferation was promoted.PART III Clinical Study of ultrasound effect in the treatment of neurodermatitis and chronic eczemaA self-controlled clinical trial was conducted in 51 patients with neurodermatitis and 51 patients with chronic eczema. The treatment group was given focused ultrasound therapy, while the control group was managed by white Vaseline, applied externally twice a day. Symptom/sign scores and total scores were evaluated at the beginning of the treatment and one month after the initiation of the treatment. Results: All symptom/sign scores and total scores were significantly decreased 1 month later.Statistically significant difference was observed between the treatment group and the control group. In the treatment group, the cure rate and improvement rate for neurodermatitis patients were 2% and 52.9% respectively ; For chronic eczema patients the cure rate and improvement rate were 8% and 54.9% respectively. The cure rate and improvement rate of the treatment group were significantly higher than those of the control group(P〈0.05〉. No statistically significant difference in efficacy was observed between the different severity(the total scores>9 or≤9)neurodermatitis and chronic eczema patients. Pruritus was significantly improved after ultrasound treatment: 90.2% of chronic eczema patients and 80.4% of neurodermatitis patients were benefited with pruritus disappearance or obvious alleviation.The side effect was zero.Conclusion: Neurodermatitis and chronic eczema could be effectively treated with focused ultrasound therapy. This approach appears to be a new physical local treatment, although further studies are still needed.更多还原